The ultimate goal of this application is the development chemical and genetic probes by high throughput screening assays for the study of endoplasmic reticulum (ER) biology and elucidating the role of the integrated ER-stress response in the pathogenesis human diseases. The Endoplasmic reticulum (ER) is the site of folding and assembly for most proteins destined for secretion or sub-cellular compartments. Reverse genetic studies indicate that abrogation of the integrated ER-stress response is lethal. ER homeostasis is therefore highly regulated and the demand for protein synthesis and the capacity of ER to fold the newly synthesized proteins must be matched. Any mismatch between the folding capacity of the ER and the demand for new proteins causes the accumulation of unfolded proteins in the ER and triggers the integrated ER-stress response, which attempts to correct the imbalance by one of three distinct, but integrated routes. Disorders of integrated ER-stress response are implicated in the pathogenesis of many malignancies, including type I and type II diabetes, cancer, inflammation, and neurodegenerative disorders. Despite this importance and the broad spectrum of malignancies associated with disorders of integrated ER-stress response, neither the chemical nor molecular genetics of the integrated ER-stress response have been studied in a systematic manner. We therefore propose to identify chemical and genetic modifiers of integrated ER stress response, which will enable in vitro and in vivo studies of the ER, and will serve as a future platform for the development of pharmaceutical agents for the therapy of human disorders in which disregulation of integrated ER-stress response is implicated. We therefore propose to take advantage of our recently developed high throughput screening (HTS) assays, which interrogate different arms of the integrated ER-stress response. Specifically we will screen libraries of chemical collections and small interfering RNAs (siRNA) to identify chemical probes and siRNAs that modify three distinct arms of integrated ER-stress response. The chemical and genetic modifiers of integrated ER-stress response will be validated in secondary and counter assays. The chemical probes identified through this effort will be critical for elucidating the role of ER-stress in the normal physiology as well as in the genesis of malignancies in which perturbations of ER- homeostasis are implicated. These probes may also serve as lead compounds for the development of ER-stress modifiers for the treatment of human disorders. Similarly, the genetic modifiers of integrated ER stress identified through this effort ma b targets for the development of novel therapeutics. PUBLIC HEALTH RELEVANCE: Mismatch between the folding capacity of the ER and demand for folding new proteins causes accumulation of unfolded proteins in the ER which then triggers integrated ER-stress response to restore the ER-homeostais. Various physiological and pathological stimuli as well as genetic mutations or other aberrations that compromise the integrated ER-stress response are associated with or implicated in the pathogenesis of many malignancies including type I and type II diabetes, inflammation, and neurodegenerative disorders including Alzheimer's and Parkinson's disease. Despite the importance of integrated ER-stress response in the pathophysiology of many disorders, there are very few chemical and genetic modifiers of the integrated ER-stress response, that we propose to remedy by screening chemical and siRNA libraries in high throughput integrated ER-stress response assay we have developed.